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Fig. S1. Microtubule cytoskeleton is required for spindle alignment. Percentage of wild-type control and MBC-treated (25 µg/ml) mto1Δ, pom1Δ, tea1Δ and tea4Δ cells showing the angle of the mitotic spindle relative to the long axis of the cell. The GFP markers used in each case are indicated.
Fig. S2. Interphase pushing MTs exert forces on the SPB. Interphase wild-type cells expressing GFP-atb2 and sid2-TOMATO as tubulin and SPB markers, respectively, were imaged every 15 seconds in multiple focal planes. One single z-section at indicated time point is shown. Arrows indicate an MT bundle contacting either cell tip. Notice that once the MT-SPB bundle hits one cell tip, the SPB is pushed in the opposite direction. The asterisk denotes a MT bundle that is being aligned with the cellular axis as it grows.
Fig. S3. Interphase MT organization and MT dynamics in repolarized orb6-25 cells. Interphase orb6-25 cells expressing GFP-atb2 and sid2-TOMATO as tubulin and SPB markers respectively were imaged every 23 seconds in multiple focal planes. Maximum projections of indicated time points are shown. The arrow indicates the position of the SPB. Scale bar: 5 µm.
Fig. S4. Intherphase MT disassembly and spindle formation in repolarized orb6-25 swollen cells. orb6-25 cells expressing GFP-atb2 and sid2-TOMATO as tubulin and SPB marker, respectively, progressing from interphase to mitosis were imaged every 15 seconds in multiple focal planes. Maximum projections of indicated time points are shown. Notice how the mitotic spindle is assembled in the exact same axis as the axis to which the interphase MT bundle attached to the SPB is aligned.
Movie 1. Correct initial spindle alignment in wild-type cells. S. pombe cells expressing the SPB marker Sid2-GFP were grown in EMM and then placed in an agar pad for the microscopic analysis. Z-series of eight focal planes with a step size of 0.5 µm were acquired every minute to track initial SPB separation. Maximum intensity image projections are shown. Note that Sid2-GFP also localizes to the actomyosin ring by the end of anaphase. Total time of movie: 37 minutes.
Movie 2. Random spindle alignment in cells treated with the microtubule-depolymerizing drug MBC. S. pombe cells expressing Sid2-GFP were grown in EMM and then placed in an agar pad containing 25 µM MBC. Z-series of eight focal planes with a step size of 0.5 µm were acquired every minute to trace initial SPB separation. Maximum intensity image projections are shown. Total time of movie: 35 minutes.
Movie 3. Random spindle alignment in mto1Δ cells. mto1Δ Sid2-GFP cells were grown in EMM and then placed in an agar pad for the microscopic analysis. Z-series of eight focal planes with a step size of 0.5 µm were acquired every minute to track initial SPB separation. Maximum intensity image projections are shown. Total time of movie: 37 minutes.
Movie 4. Initial SPB separation at mitosis occurs along the same axis as the last SPB movement during interphase. orb6-25 Sid2-GFP cells were grown at the permissive temperature (25°C) in EMM and then switched to the restrictive temperature for 4 hours to alter the cell shape. Round cells were then switched to the permissive temperature and 1 hour later placed in an agar pad for the microscopic analysis. Z-series of 12 focal planes with step size of 0.5 µm were acquired every minute to track initial SPB separation. Maximum intensity image projections are shown. Total time of movie: 4 minutes.
Movie 5. Interphase MT disassembly and the formation of the mitotic spindle. Wild-type cells expressing GFP-atb2 and sid2-TOMATO progressing from interphase to mitosis were recorded in multiple focal planes. Maximum projections of z-series are shown.
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